The creation of transgenic animals or genetically modified individuals, i.e. individuals whose genome has been modified by the permanent introduction of exogenous DNA, has assumed ever greater importance for example in gene regulation studies, for therapeutic goals and for livestock production. In particular, certain animals may be created in order to increase milk production, to be resistant to pathogens, for experimental use in the development of novel drugs or, even, for xenotransplantation.
However, to date, the creation of such animals is very complex and problematic.
Xenotransplantation, for example, belongs to a particular branch of biotechnology and medicine involved in the transplantation of cells, tissues and organs derived from individuals belonging to different species. This branch has been known for some time but encounters significant difficulties in practical application, due to problems associated with the rejection of the transplanted organs. Indeed, it is known that the immune system of the recipient body recognises the transplanted organ as extraneous, and initiates a series of reactions leading to the so-called rejection of the extraneous organ.
The aim of clinical xenotransplantation could be that of substituting a diseased human organ with organs from animals, under such conditions whereby the problem of rejection is avoided.
In order to settle this problem, along with the problems associated with the production of animals for other purposes such as those listed above, the creation of transgenic animals, or rather animals which have been genetically modified so as to carry defined genetic sequences normally not present in their genomes, has been proposed. In other words, there is an attempt to insert DNA sequences, encoding the expression of proteins which help avoid the above mentioned problems relating to rejection, into the DNA of certain target animals.
Generally, the technique most commonly used for the insertion of exogenous DNA into animal cells consists in the microinjection of exogenous DNA into the male pronucleus of a zygote. This technique, even though it has proved to be successful in mice, has not shown any degree of success in livestock, such as pigs, which are of significant interest for xenotransplantation and other biotechnologies, thereby limiting its general use.
Lavitrano et al. (1989, Cell 57, 717-723) have proposed an alternative procedure for the production of transgenic animals, know as “Sperm-Mediated Gene Transfer” (SMGT) which is based on the discovery of the capacity of sperm cells to bind and “internalise” exogenous DNA (DNA uptake), transforming them into vectors for the transmission of not just their own genetic material, but also the exogenous DNA of interest.
Advantageously with respect to other transgenic technologies, the procedure summarised above does not require any expensive equipment such as microinjectors or micromanipulators, does not require any delicate operations such as microinjection and the intervention of specialised technical staff. Furthermore, the SMGT procedure has shown itself to be generally effective for a large variety of transgenic animals with variable percentages of genes positively inserted into zygotes and expressed (Lavitrano et al. Mol. Rep. and Dev. 64:284-291, 2003).
Furthermore, the efficacy of the frequency of transfer of the exogenous genes to progeny by means of microinjection in some cases, as for instance in pigs, has not yielded satisfactory results. In addition, it has been observed that the exogenous DNA inserts randomly into the host DNA, occasionally resulting in the blockage or altered transcription of a functional gene. Indeed, the insertion of exogenous DNA sequences occurs by recombination with the host DNA in an entirely random manner, whereby it may even be inserted inside a functional gene sequence, thus completely altering its transcription. Moreover, it has been observed that the integration vectors for DNA molecules undergo the so-called gene “silencing” phenomenon, i.e. no transcription and expression of the genes inserted into the host genome has been detected.
In particular, in seeking to obviate the problem of altered transcription of host DNA, it is necessary to design DNA sequences for insertion or plasmid vectors containing said DNA very carefully and, in any case, there is always a significantly high percentage of cases where it is impossible to predict into which site in the host DNA the exogenous DNA may be inserted, and the consequences this may cause, even to the progeny once it is transmitted to them.